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Antitumour activity of the novel flavonoidOncamex in preclinical breast cancer modelsCarlos Martınez-Perez*,1, Carol Ward1, Arran K Turnbull1, Peter Mullen2, Graeme Cook3, James Meehan1,Edward J Jarman1, Patrick IT Thomson4, Colin J Campbell4, Donald McPhail3, David J Harrison2 andSimon P Langdon1

1Division of Pathology Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western GeneralHospital, Edinburgh EH4 2XU, UK; 2School of Medicine, University of St Andrews, St Andrews KY16 9TF, UK; 3Antoxis Limited, IMSBuilding, Foresterhill Health and Research Complex, Aberdeen AB25 2ZD, UK and 4EaSTCHEM, School of Chemistry, University ofEdinburgh, Joseph Black Building, Edinburgh EH9 3FJ, UK

Background: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models.We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrialtargeting might have increased potential as antitumour agents.

Methods: We studied the effect of a second-generation flavonoid analogue Oncamex in a panel of seven breast cancer cell lines,applying western blotting, gene expression analysis, fluorescence microscopy and immunohistochemistry of xenograft tissueto investigate its mechanism of action.

Results: Proliferation assays showed that Oncamex treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis,concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in theexpression of genes controlling cell cycle and apoptosis. Fluorescence microscopy showed the compound’s mitochondrialtargeting and reactive oxygen species-modulating properties, inducing superoxide production at concentrations associatedwith antiproliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showedthat Oncamex inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no signs of untoward effects on theanimals.

Conclusions: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shownantitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continueddevelopment as an anticancer agent.

Flavonoids account for the largest and most ubiquitous group ofplant secondary metabolites. They comprise seven differentsubclasses of polyphenols with a common backbone consistingof two fused rings linked to another aromatic ring (Weng andYen, 2012). Beyond their numerous roles in plant biology,flavonoids have long been identified as possessing a wide rangeof bioactivities, including protective and therapeutic effects againstcancer, cardiovascular and neurodegenerative diseases, and thushave great potential for clinical application (Romano et al, 2013).

In particular, extensive preclinical evidence has accumulatedfor their antiproliferative effects against several types of cancer,including breast (Pan et al, 2012), prostate (Brown et al, 2005),lung (Park et al, 2012) and colorectal cancers (Ko et al, 2005a).However, their use in a therapeutic context is presently hamperedby poor drug-like attributes resulting in low bioavailabilityand metabolic stability, limited cell uptake and ineffectivedelivery to important cellular compartments such as themitochondria.

*Correspondence: C Martınez-Perez; E-mail: [email protected]

Revised 17 November 2015; accepted 16 December 2015; published online 31 March 2016

& 2016 Cancer Research UK. All rights reserved 0007 – 0920/16

FULL PAPER

Keywords: breast cancer; preclinical models of cancer; ROS modulation; novel flavonoids; animal models of cancer; naturalproducts; SAR studies; xenograft models; novel antitumour agents

British Journal of Cancer (2016) 114, 905–916 | doi: 10.1038/bjc.2016.6

www.bjcancer.com | DOI:10.1038/bjc.2016.6 905

mailto:[email protected]

http://www.bjcancer.com

Previous research has reported the anticancer effect offlavonoids on breast tumours through multiple mechanisms(Martinez-Perez et al, 2014). Structural similarities to the hormone17b-oestradiol allow for interaction with the oestrogen receptors(ERs) (Limer and Speirs, 2004), although the effect is predomi-nantly beneficial due to a stronger affinity for the proliferation-inhibiting isoform ERb (Strom et al, 2004; Harris et al, 2005;McCarty, 2006). In addition, flavonoids can block the bioactivationof procarcinogens (Ciolino and Yeh, 1999; Moon et al, 2006) andact as inhibitors for oestrogen-producing and metabolisingenzymes (Sanderson et al, 2004; Rice and Whitehead, 2006) andfor the breast cancer resistance protein (BCRP), involved inthe development of multidrug resistance (Katayama et al, 2007;Tan et al, 2013).

Flavonoids exert these antitumour activities in a concentration-and time-dependent manner, and have been shown to be effectivein the treatment of numerous cancer types specifically targetingmalignant cells (Ramos, 2007). Such properties support flavonoidsas strong candidates for the development of novel anticancertreatments. Indeed, research has shown that administration offlavonoids can lead to a decrease in inflammation, proliferation,tumour size and metastasis (Limer and Speirs, 2004; Peluso et al,2013). Further potential lies in their application as re-sensitisers intumours clinically resistant to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) (Siegelin et al, 2009), radio-therapy (Yi et al, 2008), endocrine therapy (Mai et al, 2007;Tu et al, 2013) or chemotherapeutic agents like Centchroman(Singh et al, 2012).

The flavonol myricetin is a natural flavonoid with powerfulantioxidant activity that has been shown to have a therapeuticeffect in different cancers both in vitro and in vivo. It exertsapoptotic effects in combination with TRAIL (Siegelin et al, 2009)or by other mitochondrial-dependent pathways (Ko et al, 2005b),as well as inducing G2/M cell cycle arrest (Zhang et al, 2008). Wehypothesised that myricetin-derived, synthetic flavonoids withimproved antioxidant properties, specific mitochondrial targetingand optimised physicochemical properties and drug-like attributes(McPhail et al, 2009) may have enhanced potential as antitumouragents.

In this study, we characterise a small library of these myricetin-derived new chemical entities. We assessed their antitumourproperties in a panel of breast cancer cell lines, describingstructure–activity relationships (SARs) and investigatingthe mechanism of action of these compounds, including the roleof reactive oxygen species (ROS) modulation in their antitumoureffects. The potential application of such synthetic derivativesin an in vivo setting was also assessed in a human breast cancerxenograft model.

MATERIALS AND METHODS

Cell culture. Breast cancer cell lines MCF-7, MDA-MB-231,BT-549 and HBL-100 (all obtained from ATCC) were culturedin Dulbecco’s Modified Eagle Medium (DMEM) supplementedwith 10% heat-inactivated foetal calf serum (FCS) and 100 IU ml� 1

penicillin/streptomycin. These cell lines correspond to differentmolecular subtypes of breast cancer, with hormone-dependentMCF-7 expressing ERs (ERþ ) and MDA-MB-231, BT-549 andHBL-100 being characterised as triple-negative cell lines, lacking inreceptors for oestrogen, progesterone and human epidermalgrowth factor (ER� PR� HER2� ) and, hence, hormone-independent. The LCC1, LCC2 and LCC9 (hormone-independentcells established by derivation of selected subpopulations of MCF-7cells (Brunner et al, 1993b, 1997; Thompson et al, 1993)) werecultured in phenol red-free DMEM supplemented with 5% heat-

inactivated FCS charcoal-stripped of steroids. Cells were incubatedat 37 1C in a humidified atmosphere containing 5% CO2. Cellswere grown to confluence with periodic medium changes andcollected by brief incubation with trypsin/ethylenediaminetetraa-cetic acid solution. All cell lines were authenticated by shorttandem repeat profiling undertaken by the Public Health England(Salisbury, UK) in December 2014.

Sulforhodamine B assay. Cells (500–2000 cells per well, depend-ing on doubling time) were typically plated in 96-well microplates,and medium changed to 2.5% FCS DMEM after 24 h, followingpreliminary experiments that showed an improved effect ofdrugs for lower serum concentrations (Supplementary Figure 1).Cells were treated after a further 24 h with a library of eightcompounds, including myricetin and lead compounds AO-1530and Oncamex (Table 1). Treatments included a range ofmicromolar concentrations between 0.01 and 100 mM for allcompounds. Each experiment was repeated at least once.

After treatment cells were fixed with 50 ml per well of cold 25%trichloroacetic acid for 1 h at 4 1C and washed 10 times with H2O.After drying, plates were stained for 30 min with 50 ml per well of0.4% (w/v) sulforhodamine B (SRB) (in 1% acetic acid) and washedfour times with 1% acetic acid. Protein-bound SRB was solubilisedin 150 ml per well of 10 mM Tris solution (pH 10.5), followed bymeasurement of optical density (OD) at 540 nm in a BP800Microplate Reader (Biohit, Helsinki, Finland). Results wereprocessed, subtracting average values of blanks and day 0 controlsand normalising to untreated controls.

Fluorescence microscopy. Cells were grown on coverslips andtreated with Oncamex for 15 min, 1 or 6 h at 37 1C, adding 25 nM

Mitotracker Deep Red (Life, Eugene, OR, USA) for the last 30 min(or 15 min for the shortest incubation). Cells were fixed in 4%parafarmoldehyde for 45 min, washed three times in phosphate-buffered saline (PBS) and allowed to air-dry before mounting onSuperfrost Plus microscope slides (Thermo Scientific, Braunsch-weig, Germany) using ProLong Gold antifade mountant withDAPI (40,6-diamidino-2-phenylindole, Life, Bleiswijk, Nether-lands). Oncamex possesses fluorescent properties, with a stablesignal measurable at 550 nmEXC/570 nmEM. Cells were visualised,captures obtained and analysed using a PM-2000 AQUA(Automated Quantitative Analysis) system (HistoRX, New Haven,CT, USA) and an Axioplan 2 fluorescence microscope (Zeiss,Cambridge, UK).

The production of ROS was detected by fluorescence micro-scopy. The method above was followed with additional staining ofcells with mitochondrial probes following the manufacturers’guidelines, incubating with 10 mM MitoPY1 (Tocris, Bristol, UK) or2.5 mM MitoSOX (Life) to detect mitochondrial production ofhydrogen peroxide and superoxide, respectively.

Cell spotting. A total of 105 MCF-7 cells per well were plated intriplicate in six-well plates. After treatment and incubation, cellswere trypsinised and centrifuged for 5 min at 2000 r.p.m. Resultingpellets were resuspended in 100 ml of FCS and transferred toCytofunnels EZ Single (Fisher, Braunschweig, Germany) mountedon Superfrost Plus slides (VWR International, Leuven, Belgium) tobe spun for 3 min at 500 r.p.m. in a Cytospin 4 Cytocentrifuge(Thermo Fisher, Chesire, UK). Slides were allowed to air-dry andstained using the Reastain Quick Diff kit (Reagena, Toivala,Finland), to fix and dye both protein and DNA. After rinsing andair-drying, cells were observed on an Olympus BX51 microscope(Olympus, Hamburg, Germany) and images were captured usingthe software package Q-Capture Pro (Q Imaging, Surrey, BC,Canada).

Plate-based assays. The CellTox Green Cytotoxicity and ApoTox-Glo Triplex assay kits (Promega, Madison, WI, USA) were used tomeasure cytotoxicity at different timepoints and to assess the

BRITISH JOURNAL OF CANCER Oncamex as antitumour agent in breast cancer

906 www.bjcancer.com | DOI:10.1038/bjc.2016.6


mechanism of action of the drugs studied, respectively. In all, 104

cells per well were plated in 96-well microplates (black walls forCellTox assay and white for ApoTox), changing the medium tophenol red-free DMEM after 24 h. The protocols were carried outfollowing the manufacturer’s instructions, and fluorescence andluminescence were measured in a Labsystems Fluoroskan AscentFL (Thermo, Vantaa, Finland) plate reader.

Cell lysates. In all, 3� 106 MCF-7 cells per dish were plated in140-cm2 Petri dishes. After treatment, cells were washed in PBSand incubated for 10 min on ice in 400 ml of lysis buffer (50 mM

Tris, 5 mM EGTA and 150 mM NaCl) containing CompleteProtease Inhibitor Tablet (Roche, Mannheim, Germany; 1 tabletper 10 ml), 1 : 100 of phosphatase inhibitor cocktails 2 and 3(Sigma, St Louis, MO, USA), 1 : 200 aprotinin (Sigma) and 1 : 100

Table 1. Library of novel flavonoids screened for their antitumour properties

Compound Structure Characteristics/propertiesMyricetin Naturally occurring flavonoid identified as particularly powerful antioxidant

AO-1530 Myricetin-based novel flavonoid, mitochondria-targeted and with active redox properties.Non-redox-active OH groups removed and a decyl chain similar to the one found in vitaminE has been added to improve permeability and targeting

Oncamex (R1)

AO-1486 (R2)

AO-1487 (R3)

AO-594 (R4)

Bi-methoxylated second-generation analogue of AO-1530

Same backbone as AO-1530, but nonspecific targeting

Same backbone as AO-1530, but nonspecific targeting

Same backbone as AO-1530, but weaker specificity for the mitochondrial compartment

AO-155-179 Similar backbone to that of AO-1530 but one of the fused rings has been removed, leaving a lessflavonoid-like structure

AO-714A Fully blocked redox activity

AO-594 Same backbone as AO-1530, but nonspecific targeting

Abbreviations: R1¼ (CH2)9-CH3; R2¼ (CH2)3-NH-(CH2)4-NH2; R3¼CH2-(C6H8)-NH2.HCl; R4¼no radical; R5¼ (CH2)10-CH3. The addition of different moieties and radicals granted distinctiveredox potential and intracellular targeting to analogues in a library of seven novel, myricetin-derived flavonoids.

Oncamex as antitumour agent in breast cancer BRITISH JOURNAL OF CANCER



Triton X (Sigma). Lysates were centrifuged at 13 000 r.p.m. at 4 1Cfor 6 min. Supernatants were recovered and stored at � 70 1C.

Bicinchoninic acid assay. Protein concentration in cell lysates wasdetermined by bicinchoninic acid (BCA) assay. Bovine serumalbumin (BSA, G Biosciences, St Louis, MO, USA) was used asprotein standard, preparing serial dilutions (0–1000 mg ml� 1) indistilled water (dH2O), while aliquots of cell lysates were alsodiluted 1 : 10 in dH2O. A volume of 1 ml of a 1 : 50 coppersulphate : BCA solution was added to 50 ml of each protein solutionin borosilicate glass tubes. Tubes were incubated at 60 1C for15 min before cooling briefly and dispensing replicates of eachsolution to a 96-well microplate. The OD at 540 nM was measuredin a BP800 Microplate Reader (Biohit) and protein concentrationin each lysate was extrapolated from BSA dilutions standardcurves.

Electrophoresis and western blot. Cell lysates (40 mg of protein in1 : 4 volume of 5� loading buffer (5% SDS, 25% 2-mercaptoetha-nol, 50% glycerol, 0.02% bromophenol blue and 0.04 M Tris)) weredenatured at 60 1C for 1 h and electrophoretically separated bypolyacrylamide gel electrophoresis on Mini-gel equipment(BioRad, Hemel Hempstead, UK). Prestained protein marker,Broad Range (7–175 kDa, New England BioLabs, Ipswich, MA,USA), diluted 1 : 3 in 1� loading buffer, was used as marker.

Proteins were transferred to a polyvinylidene fluoride mem-brane for 90 min at a constant voltage of 100 V and in cold, stirredtransfer buffer (25 mM Tris and 19.2 mM glycine). After blockingfor 1 h at 4 1C in BB : PBS (1 : 1 dilution of Odyssey Blocking buffer(Li-Cor, Lincoln, NE, USA) in PBS), membranes were incubatedwith mouse anti-cleaved PARP (poly(ADP-ribose) polymerase;1 : 8000 dilution, Cell Signaling, Hitching, UK) and rabbit anti-human b-tubulin (1 : 6000 dilution, Abcam, Cambridge, UK)antibodies overnight at 4 1C. Staining with secondary goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680CWantibodies (both 1 : 10 000 dilution, Li-Cor) was followed byscanning on Li-Cor Odyssey Scanner (Li-Cor).

Analytical electrochemistry. To prepare solutions of analytes,0.5 mg of solid sample was suspended in 1 ml of 100 mM

tetrabutylammonium hexafluorophosphate dissolved in acetoni-trile (MeCN), and the suspension was treated with ultrasound in awater bath (40 1C) for 30 min. Cyclic voltammograms wereacquired using a Autolab PGStat (Eco-Chemie, Utrecht, Nether-lands) at a scan rate of 100 mV s� 1, using only the tenth and finalscan unless otherwise stated, to estimate the reduction potential ofthe analytes. A saturated calomel (Hg2Cl2) electrode was used as areference and a fine platinum gauze (0.1 mm wire, 1 cm2) as acounter electrode. A straight platinum wire (1 mm diameter) wasused as a working electrode, and the experiments were carried outunder a blanket of argon.

RNA processing, microarray hybridisation and data analysis.Raw and normalised gene expression files are available from theNational Center for Biotechnology Information Gene ExpressionOmnibus (Barrett et al, 2005) under the accession numberGSE70949.

Ten samples were collected comprising five breast cancer celllines (MCF-7, MDA-MB-231, LCC1, LCC2 and LCC9) in twodifferent conditions: untreated vehicle control (DMSO) and treatedcells (6 h in 10 mM Oncamex). For this, 3� 106 cells per cell linewere treated, trypsinised and stored at � 70 1C. The RNA wasextracted using the Qiagen RNeasy Mini kit (Qiagen, Hilden,Germany), amplified and labelled using the Ambion IlluminaTotalPrep RNA Amplification kit (Life, Carslbad, CA, USA)(in both steps as per the manufacturers’ instructions) andhybridised to HumanHT-12 v4 Illumina BeadChips (Illumina,Cambridge, UK). Arrays were scanned using an Illumina iScan(Illumina).

Raw gene expression files were log2-transformed and quantile-normalised using the lumi Bioconductor package (Du et al, 2008),mapped to Ensembl gene identifiers and detection-filtered usingre-annotation and pre-processing approaches previously described(Turnbull et al, 2012). Differential gene expression analysis wasperformed using pair-wise rank products (Breitling et al, 2004)between control and treated groups. Functional enrichmentanalysis of differentially expressed genes was performed usingDAVID (Database for Annotation, Visualization and IntegratedDiscovery) Bioinformatics Resources 6.7 (Huang et al, 2009a, b).Heatmaps of differentially expressed genes belonging to clustersenriched for cell cycle and apoptosis were generated using log2 foldchange expression values calculated between treated and controlconditions for each cell line. Heatmaps were generated using TM4microarray software suite’s MultiExperiment Viewer (Saeed et al,2003, 2006) and genes were ordered by Euclidean distance. Genesbelonging to the apoptosis cluster were differentiated into pro- andanti-apoptosis clusters using Qiagen’s custom referenced apoptosisPCR array literature (references therein).

Xenograft experiments. The xenograft studies were undertakenunder a UK Home Office Project Licence in accordance with theAnimals (Scientific Procedures) Act 1986, and studies wereapproved by the University of Edinburgh Animal Ethics Commit-tee. The MDA-MB-231 xenografts were implanted subcutaneouslyinto the flanks of adult (48 weeks) female CD-1 immunodeficientmice (Charles River Laboratories, Tranent, UK), using 10xenografts per experimental group of 6 mice, implanted in oneor both flanks. Treatment was started when the mean tumourvolume reached 0.25 cm3 (day 0) and mice were treated dailyintraperitoneally with Oncamex (25 mg kg� 1 per day) or withsolvent control (10% DMSO in saline) on days 0–4 and 7–11.Changes in tumour size over 14 days were measured using Verniercallipers and volumes calculated (V¼ l�w2/2). Changes in meanbody weight were recorded every 2 days over 14 days. The initialdose selection of 25 mg kg� 1 per day was based on the similarity ofthis structure to another compound for which 25 mk kg� 1 per dayhad been a safe dose. This dose was confirmed to be safe inan initial dose-testing experiment and no untoward effectswere noted.

Immunohistochemistry. Xenograft tissue was collected, fixed informalin and embedded in paraffin. Sections were dewaxed inxylene for 5 min and washed in alcohol and water beforeincubating in heated antigen retrieval solution (0.1 M sodiumcitrate and 0.1 M citric acid, pH 6). Slides were washed in PBS,incubated in 3% dH2O2 for 10 min and washed again in PBST(0.1% Tween-20 PBS) before incubating for 10 min in TotalProtein Block (Dako, Ely, UK). Sections were incubated for 1 h inmouse monoclonal anti-Ki-67 antibody (1 : 300 dilution, Dako),followed by 30 min in Envision labelled polymer (Dako) and10 min in DAB (1 : 50 dilution in buffer, Dako), with washes inPBST between each step. Finally, slides were counterstained inhaematoxylin for 1 min and taken through graded alcohols toxylene before mounting in DPX mountant medium (Sigma-Aldrich, Dorset, UK).

Immunohistochemistry (IHC) scores were calculated by count-ing average positively stained cells across sections for each of the 10xenografts for treated and control groups, using the average ofcalculations by three users to ensure unbiased estimations.Percentage of viability was assessed using the image-processingpackage Image J (NIH, Bethesda, MD, USA) to measure the viableareas in each section.

Other materials. Novel flavonoids tested (Table 1) were suppliedby Antoxis Limited (Aberdeen, UK) from their library ofproprietary compounds (Caldwell et al, 2007). They were customsynthesised and their purity was ascertained to be 495% by liquid




chromatography mass spectrometry and nuclear magnetic reso-nance. Unless otherwise stated, any other reagents and solventswere of analytical grade from Sigma-Aldrich and were usedwithout further purification.

Statistical analysis. For analysis of SRB and other plate-basedassays, all experiments were repeated at least once, and sixtechnical replicates were used to calculate means and s.d. Resultswere processed, subtracting average values of blanks and day 0controls and normalising to untreated controls. Cell proliferationcurves were fitted to a model for sigmoidal regression using theExcel package Fit Designer 2D (IDBS, Guildford, UK), excludingoutliers outside of a 95% confidence interval, for the calculation ofhalf maximal inhibitory concentration (IC50) values.

For statistical analysis of xenograft and IHC results, Prism 6(GraphPad Software, La Jolla, CA, USA) was used to comparecontrol and treated groups using unpaired t-test.

RESULTS

Novel flavonoid Oncamex exerts a potent antitumour effect inbreast cancer cell lines. Among the eight compounds tested(Table 1) in a panel of SRB assays, AO-1530 and its second-generation methoxylated analogue Oncamex showed the strongestantiproliferative effects (Figure 1A and B). Oncamex was the mostpotent myricetin derivative of the series tested and exerted similarantiproliferative responses in MCF-7, BT-549, MDA-MB-231 andHBL-100 cells. For hormone-independent cell lines LCC1, LCC2and LCC9, the two lead compounds were still more effective thanmyricetin although the overall effect was weaker.

Oncamex’s IC50 values were between 4- and 140-fold lower thanthose of myricetin in all cell lines, while the potency and IC50

values of the other analogues were more variable (Figure 1C and D).Analogues AO-714A and AO-155-179 typically exerted antiproli-ferative effects closer to those of the lead compounds, whereasother analogues exerted weaker effects, with IC50 values closer to,or even higher, than those of myricetin.

Oncamex specifically targets the mitochondrial compartment,with rapid delivery and stable accumulation. Drug uptake andintracellular location was assessed by microscopic visualisation.Images obtained demonstrated a specific targeting of themitochondrial compartment by Oncamex in the model breastcancer cell line MDA-MB-231 (selected for its better adherencewhen grown on coverslips; Figure 2). The compound wasco-localised with Mitotracker (and absent from nuclei andcytoplasm) as early as 15 min after treatment and was still retainedin the mitochondrial compartment after 6 h. Previous work inanother cancer cell line has shown the ability of AO-1530 to targetthe mitochondria, whilst other analogues with weaker antiproli-ferative effects have less specific intracellular localisation(Supplementary Figure 2).

Oncamex exerts its antitumour effect through induction ofcytotoxicity and apoptosis. Microscopic observation of MCF-7cells showed that treatment with Oncamex induced substantialchanges after 24 h, including a reduction of cell density anddivision, alterations in nuclear morphology and appearance ofapoptotic cells (Figure 3A–D). By 72 h after treatment thesechanges were generalised, with abundant apoptotic, phagocytisedand dead cells. These results suggested the involvement ofapoptosis in Oncamex’s mechanism of action.

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43.3088 12.2097 2.4504

19.0746 2.7173 0.7424

79.9474 24.5072 5.1804

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Figure 1. Antitumour effect of Oncamex on breast cancer cell lines. The antiproliferative effect of a library of novel flavonoids was first assessedthrough SRB assays on a panel of seven breast cancer cell lines. Cells were treated for 4 days with drug concentrations in the 0.01–100mM rangeconcentration in all treatments. Results were comparable in all models, including MCF-7 (A) and MDA-MB-231 cells (B), with second-generationanalogue Oncamex showing the strongest anticancer properties, markedly greater than those of myricetin. Analysis of concentration–responsecurves obtained from SRB assays allowed for the calculation of IC50 values for myricetin and the lead compounds (C, D), which reflected thestronger potency of Oncamex.




SRB assays on cells exposed to different lengths of treatment (8,16, 24, 48, 72 and 96 h) provided an insight into the timing of theanticancer effects observed (Supplementary Figure 3). Resultsshowed that besides the more potent effect of Oncamex over AO-1530, the former also exerts a more rapid effect, inducing asignificant reduction in cell density after 8 h comparable to thelevels observed after 96 h, whereas AO-1530’s effect was weakerand more delayed, not affecting cells until 24 h into treatment.CellTox plate assays showed that Oncamex produced an increasein cytotoxicity by 8 h after treatment in all cell lines (or earlier insome of them), reaching levels between 2- and 10-fold greater thanthe baseline signal by 24 h (Figure 3E).

Further investigation of Oncamex’s mechanism of action usingApoTox multiplex assays showed that treatment of MCF-7, MDA-MB-231, HBL-100 and BT-549 cells with micromolar Oncamexconcentrations for 8 h induced concentration-dependent, inverselycorrelated changes in cytotoxicity and cell viability, together withcaspase-3/-7 activation, consistent with apoptosis (Figure 3G).

Induction of apoptosis was also measured by western blottingdetection of PARP cleavage. Treatment with micromolar concen-trations of AO-1530 or Oncamex led to cleavage of PARP.Oncamex exerted a more rapid effect, with cleaved PARP beingdetectable after 8 h, whereas 24-h incubation with AO-1530 wasrequired for PARP cleavage to occur (Figure 3F).

ROS modulation is linked to Oncamex’s properties. Oncamexdisplays an active redox profile. Analysis of electrochemical activityby cyclic voltammetry showed that Oncamex undergoes areversible reduction, with a midpoint potential of þ 145 mV vsnormal hydrogen electrode (Supplementary Figure 4).

To assess the role of ROS modulation in Oncamex’s antitumoureffect, ApoTox assays were repeated with the addition of a 30-minpre-incubation stage using an antioxidant agent (5 or 10 mM

N-acetyl cysteine, NAC). Results with both model cell lines used

(MCF-7 and MDA-MB-231, selected to include both an ER-positive and a triple-negative cell line, respectively) showed pre-treatment with 10 mM NAC caused a partial, but not complete,blockage of the cytotoxic and apoptotic signals induced bytreatment with Oncamex (Figure 3H).

To investigate the effect of Oncamex on ROS production, MDA-MB-231 cells (selected for their better adherence when grown oncoverslips) were stained with the novel fluorescent probes MitoPY1or MitoSOX for the specific detection of mitochondrial hydrogenperoxide (mH2O2) and superoxide (mSO), respectively. The signalgenerated by MitoPY1 was not intense enough to allow forsensitive quantification, but provided qualitative results in the formof bright specks localised in the mitochondrial department.The induction of mH2O2 was observed 1 h after treatmentwith 0.3mM Oncamex but no significant signals arose afterlonger incubations (Figure 4A). MitoSOX results showed aquantifiable, significant increase in mSO production in cellstreated with higher concentrations of Oncamex for longertreatment times (Figure 4B and C).

Oncamex induces gene expression changes related to cell cycleand apoptosis regulation. Results from microarray experimentsshowed that 6-h treatment with Oncamex altered the expressionprofile of genes related to cell cycle and apoptosis (Figure 5). Genesinvolved in cell cycle regulation were downregulated by treatmentin all cell lines studied. These include genes encoding proteinswith well-known biological functions such as cyclins (encoded byCCND1, CCNF or CCNB1), regulators of proliferation (AURKAand MKI67) and other cell division-promoting proteins (CDC20,MCM5 or MCM3).

Functional enrichment analysis showed that Oncamex inducesdifferent effects in two clusters of apoptosis-related genes.Pro-apoptotic genes were upregulated by treatment, includinggenes encoding apoptosis-inducing proteins (such as BNIP3,

15 min 1 h 6 h

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Figure 2. Intracellular localisation of Oncamex. Fluorescence microscopy on MDA-MB-231 cells treated with Oncamex demonstrated the rapiddelivery of the drug and its localisation within the mitochondrial compartment, as shown by the overlap of the drug’s fluorescent signal with thatof Mitrotracker Deep Red (visualised as an orange–yellow signal resulting from the overlap of red and green fluorescences). The compound isdelivered to the mitochondria as early as 15 min after the treatment and accumulates over time, retaining its organelle-specific targeting after 6 h.Visualisation of the cells at higher magnifications showed the change in cell morphology after 6 h, indicative of the induction of apoptosis.




BNIP3L or CRADD) and caspases (CASP7). Anti-apoptotic geneswere downregulated, including decreased expression of genesinvolved in apoptosis inhibition (DFFA, BCL2, BIRC6 or BIRC5) orsurvival and proliferation (IL10).

Oncamex inhibits tumour proliferation and viability in a mousein vivo model. Results from a first in vivo model in miceimplanted with MDA-MB-231 xenografts (selected for the betterability of these cells to grow as xenografts) showed that treatmentwith Oncamex (25 mg kg� 1 per day) had a significant effect(Po0.05), inhibiting tumour growth as indicated by changes inxenograft volume compared with untreated mice (Figure 6A).Treatment did not entail significant changes in mean body weight(o4% loss over 20 days) (Figure 6B).

IHC processing of collected xenograft tissue identified asignificant decrease in Ki-67 expression, from 60% in controls to36% in mice treated with Oncamex (Figure 6C–F), suggesting thatthis compound inhibits cell proliferation in vivo. Similarly, thepercentage of viable areas was significantly reduced from 56 to 37%(Figure 6C–E and G).

DISCUSSION

In this study, we have identified several novel flavonoids withgreater potency than myricetin when assessed in a panel of sevenbreast cancer cell lines. AO-1530, a synthetic analogue of myricetin

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Figure 3. Cytotoxic and apoptotic effects of Oncamex. Initial evidence on the mechanism of action of Oncamex was obtained from study ofbreast cancer cell line models MCF-7 and MDA-MB-231. Microscopic visualisation of MCF-7 cells treated with 30mM Oncamex showed a reductionin cell density, changes in cell morphology and the appearance of apoptotic and dead cells after 24 h (A–C), which was generalised by 72 h aftertreatment (D). CellTox Green plate assays supported preliminary results by direct measurement of the induction of cytotoxicity in treated MDA-MB-231 cells, peaking 8 h after treatment (E). Western blotting was used to measure PARP cleavage in treated MCF-7 cells (F): Oncamex seemedto induce apoptosis faster, with PARP cleavage occurring after 8 h treatment with the highest concentrations, whereas 24-h incubation wasrequired with AO-1530. The 24-h incubation with the highest concentrations seemed to induce further cell degradation, preventing proteindetection. Apoptosis was confirmed by an additional method using ApoTox plate assays, which reported an dose-dependent increase incytotoxicity and apoptosis, inversely proportional to cell viability (G). These antitumour effects were partially blocked by 30 pre-treatment with10 mM of the antioxidant NAC (H).




in which the non-redox-active OH groups on the A-ring of theflavonoid have been removed and a decyl chain has been added toimprove cell membrane permeability (analogous to the function ofthe chain in vitamin E), had previously been identified as amore potent antioxidant than myricetin (McPhail et al, 2009).Results from SRB assays have indicated that AO-1530’s strongantiproliferative properties are surpassed by Oncamex, a second-generation bi-methoxylated analogue.

We aimed to identify the SAR properties contributing tothe stronger potency of these novel analogues. The library ofcompounds studied included molecules specifically designed toachieve distinct intracellular targeting and effective redox proper-ties. One of the defining characteristics of Oncamex is the inclusionof two methoxy moieties in its structure. Previous research hassuggested contrasting effects of methoxy substitutions in chemicalentities: it has been reported that they may have unfavourablesteric effects, compromising redox-modulating and cytochromeP450 (CYP1)-inhibitory capabilities (Heim et al, 2002; Arroo et al,2009), while the extent of the BCRP-inhibitory properties offlavonoids also depends on the number and location of thesemodifications (Katayama et al, 2007; Pick et al, 2011; Tan et al,2013). Nevertheless, the enhanced potency of Oncamex is mostlikely the result of methoxylation leading to improved pharmaco-kinetic properties and increased stability: methoxylated com-pounds are less prone to modifications such as glucuronidationand sulphation, and are thus more chemically and metabolicallystable (Androutsopoulos et al, 2010). Added to improved uptakeand membrane transport, such alterations may provide thesecompounds with increased bioavailability (Walle, 2007; Arroo et al,2009). Moreover, it has been reported that upon delivery,

methoxylated compounds are targeted by tumour-specificO-demethylases that provide free hydroxyl groups and hence anincrease in redox properties (Androutsopoulos et al, 2008; Arrooet al, 2009). Therefore, it seems reasonable that chain-bearing,methoxylated novel flavonoids could make promising candidatesas potential chemotherapeutic agents, providing improved phar-macological attributes, including cancer-specific activation.

Although Oncamex was the most potent compound in thisseries, the effect of the other analogues was more variable.Interestingly, the fully methoxylated molecule AO-714A showed apotent effect with variable but generally low IC50 values. This alsosupports the notion that flavonoids may not require freehydroxyl groups to be active anticancer agents due to theirpropensity to undergo demethylation in vivo by O-demethylases(Arroo et al, 2009). Similar to Oncamex, AO-714A presentsmultiple methoxylations that would improve its bioavailability andthese are located in the 30, 40 and 50 positions, reported as structuralfeatures that significantly increase the BCRP-inhibitory activity offlavonoids (Katayama et al, 2007; Tan et al, 2013). Theseobservations suggest that different mechanisms such as inductionof oxidative phosphorylation-independent cell death (as previouslyreported with myricetin (Ko et al, 2005b)) could also be relevant totheir anticancer effect. Finally, other analogues expected to have aredox potential comparable to that of AO-1530 but with no specifictargeting for mitochondria showed lower potency, highlighting theimportance of targeted drug delivery.

Overall, these results indicate that particular SARs are relevantfor the application of flavonoids to cancer treatment. Theantitumour effect of these novel molecules is most likely the resultof a combination of different structural traits and properties,

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Figure 4. Effect of Oncamex on production of mitochondrial ROS. The production of ROS in MDA-MB-231 cells exposed to Oncamex in differenttreatment conditions was assessed using mitochondrial, species-specific fluorescent probes for microscopic visualisation of production of differentspecies in situ. MitoPY1 produced a signal too weak for reliable quantification, but provided qualitative results showing an increase in mH2O2 aftershort incubations with 0.3mM Oncamex (A), visible as bright specks in the mitochondrial compartment similar to the ones observed in the positivecontrol treated with 100mM H2O2. Measurement of MitoSOX reported a quantifiable, significant increase in mSO 6 h after treatment with 30 mM

Oncamex (B), as supported by statistical analysis of measurements (C). P-values from unpaired t-test: **Po0.01; ***Po0.001; ****Po0.0001.Where not shown P40.05 (nonsignificant).




including optimum redox potential and structural resemblance tothe flavonoid backbone, which has been shown to allow for a widerange of molecular interactions for different polyphenols(Martinez-Perez et al, 2014). In addition, the substitution withelectron-donating moieties, methoxylated residues and mitochon-drial targeting provide significantly stronger antitumourproperties.

Oncamex exerts an antiproliferative effect on cancer cells andhas been found to induce apoptosis, as suggested by thedevelopment of responses as soon as 8 h after treatment bydetection of caspase activation and PARP cleavage. Results fromthe panel of breast cancer cell lines studied suggest that theantitumour effect of Oncamex is independent of ER status, astriple-negative cell lines MDA-MB-231, BT-549 and HBL-100 cellsshowed susceptibility similar to that of ER-positive cells.This suggests that Oncamex’s effect does not rely on modulationof hormonal signals and thus may be applicable to cancers of amore varied nature. We observed that LCC cell lines, whichare ER-positive but hormone-independent MCF-7 variants,are less sensitive to Oncamex, but this is most likely due to thelower baseline proliferation rate of LCC cells (Brunner et al,1993a, b, 1997).

The cytotoxic and apoptotic signals induced by Oncamex werepartially blocked by pre-incubation with an antioxidant, suggestingthat the compound’s redox activity, studied by electrochemicalanalysis, is at least partially involved in the induction of these

responses through ROS modulation. The production of ROS wasdetected by fluorescence microscopy using species-specific probesto obtain in situ visualisation. Results reported the induction ofdifferent mitochondrial ROS production in a concentration- andtime-dependent manner. Whereas mH2O2 was produced shortlyafter treatment with nanomolar concentrations of Oncamex, mSOwas produced after 6 h treatment with higher concentrations, in thesame timeframe and concentration range linked to Oncamex’santitumour properties. The redox profile for Oncamex showed thatit undergoes a reversible reduction with a midpoint potential ofþ 145 mV, a value substantially more positive than the physiolo-gical resting redox potential in either cytoplasm or mitochondria(Auchinvole et al, 2012; Mallikarjun et al, 2012). This supports thenotion that although aspects such as kinetics and biostability mayaffect its reactivity, Oncamex would most likely be found in itsreduced form in the mitochondrial compartment and thus be ableto donate an electron to oxygen to form the superoxide radicalanion. Hence, results obtained from the study of mitochondrialROS suggest that production of superoxide in the mitochondrialcompartment might be associated with the antitumour effectsexerted but also support the existence of a biphasic effect,previously reported for polyphenols such as flavonoids (Ramos,2007), by which different, possibly opposing effects might beexerted by a compound depending on a fine concentration balance.

Gene expression analysis was carried out to study the effect ofOncamex on breast cancer cell line models at gene expression level.

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Figure 5. Modulation of gene expression by treatment with Oncamex. Heatmaps based on log2 fold changes between control and treated (6 h10mM Oncamex) summarising changes in gene expression in cell cycle and apoptosis function groups. Red and green represent high and low log2gene expression fold changes, respectively. Genes are ordered by Euclidean distance. Representative examples of well-characterised genes ineach pathway, which are up- or downregulated the most on treatment, are enlarged in panels to the right and their position in the overall heatmapis indicated by colour-coded markers.




Cells were exposed to treatment levels that had been shown to exertchanges in proliferation and cell death, as well as to induce changesin ROS signalling. Results supported the antiproliferative and pro-apoptotic effects previously measured using cytotoxicity assays,western blotting and different plate assays. This suggests that therapid delivery of Oncamex to the mitochondria is also translatedeither directly or indirectly into regulation of these pathways atgene expression level. Importantly, Oncamex induced largely thesame effect across different cell lines despite their inherentbiological differences.

The ROS status has been established as a major regulator in thedevelopment and advancement of cancer and, as the site of cellrespiration, the mitochondrial compartment is the main source ofROS-linked signalling. Hence, a number of different mechanismscould be altered by the effect of these ROS-modulating drugs in themitochondria. Given the versatility of flavonoids as anticanceragents, these results show that while Oncamex’s promise as anantitumour agent has been demonstrated, its mechanism of actionis probably complex and further study will be required for a betterunderstanding.

Oncamex demonstrated significant growth-inhibitory activity inthe MDA-MB-231 breast cancer xenograft model at a dose levelthat was not associated with any indications of toxicity. Theproliferative marker Ki-67 was reduced by treatment with the drugconsistent with an antiproliferative effect. Further studies are nowrequired to observe whether other breast cancer xenografts are

sensitive to this drug and to further study the pharmacokinetic andpharmacodynamic properties of this agent.

In conclusion, we have shown the potential of the proto-typic novel compound Oncamex as an antitumour agent. Thissuggests that myricetin’s natural scaffold can be modified toenhance its activity and shows the potential for mitochondrialtargeted, redox-active molecules in cancer therapy. The resultsreported in this study have provided evidence of the anticancereffect of Oncamex in in vitro cell culture models, as well aspreliminary antitumour activity in an in vivo xenograft modelin mice.

ACKNOWLEDGEMENTS

We are grateful to Professor Robert Clarke for the use of the LCC1,LCC2 and LCC9 cell lines; to Sonya Uddin and ChrysiXintaropoulou for their help with the gene extraction procedures;to Paul Perry for his assistance with microscopy; and to HelenCaldwell and Elaine McLay for sectioning of formalin-fixed andparaffin-embedded tissues. We thank SULSA (Scottish UniversitiesLife Science Alliance) for supporting this project through a SULSABioSkape Industry PhD Studentship and Antoxis Limitedfor providing additional funding. We also thank the SeventhFramework Programme of the European Union (METOXIAproject; HEALTH-F2-2009-222741) for support.

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Figure 6. Effect of Oncamex on xenografts in an in vivo mice model. Treatment of mice implanted with MDA-MB-231 xenografts with Oncamex(25 mg kg�1 per day, treating for 4 consecutive days with a 2-day rest in between) showed to exert a significant growth-inhibitory effect incomparison to untreated animals (A). Mean body weight was registered every second day for 20 days (B). Ki-67 activation was measured byimmunohistochemistry in controls (C) and xenografts treated with Oncamex (D). Statistical study reported a statistically significant decrease inexpression of the proliferation-linked protein as reported unpaired t-test analysis (E, F). Image analysis with the open-access processingprogramme ImageJ also showed a statistically significant reduction in the percentage of viable areas in the tissue (E, G). P-values from unpaired t-test: *Po0.05; **Po0.01; ***Po0.001; ****Po0.0001. Where not shown P40.05 (nonsignificant).




CONFLICT OF INTEREST

DM and GC are employees of Antoxis Limited. The remainingauthors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

CM-P, CW, DJH, DM and SPL conceived of the study andparticipated in its design and coordination, and helped to draft themanuscript; CM-P, AKT, JM and EJJ participated in the geneextraction and IHC procedures; PM and CW assisted with cellculture; GC assisted with microscopy; PITT and CJC assisted withanalytical electrochemistry and participated in cyclic voltammetryexperiments; DM, PITT and CJC provided guidance on chemistryand ROS biology; CM-P and AKT carried out the gene expressionanalysis; SPL carried out the in vivo experiments; all authorscontributed to and approved the final manuscript.

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